Western blot analysis

Western blotting was conducted as described previously using whole cell lysates (31). Lysate of transfected cells was collected 4 days after siRNA transfection and 2 days after GRHL2 overexpression. Cell lysate of untransfected normal and lung cancer cell lines was collected at ~80% confluency. The primary antibodies used were rabbit anti-GRHL2 (cat. no. HPA062839; Merck KGaA; 1:500), mouse anti-E-cadherin (cat. no. 610181; BD Biosciences; 1:1,000), mouse anti-vimentin (cat. no. HPA001762; Merck KGaA; 1:500), rabbit anti-AKT (pan; cat. no. 4691; Cell Signaling Technology, Inc.; 1:1,000), rabbit anti-phosphorylated (p-)-AKT (ser473; cat. no. 4060; Cell Signaling Technology, Inc.; 1:1,000), rabbit anti-p44/42MAPK (Erk1/2; cat. no. 9102; Cell Signaling Technology, Inc.; 1:1,000), rabbit anti-p-p44/42MAPK (Erk1/2; cat. no. 4370; Cell Signaling Technology, Inc.; 1:1,000) and mouse anti-β-actin (cat. no. A2228; Merck KGaA; 1:2,000). The β-actin protein level was used as a control for protein loading. The primary antibodies were incubated at 4°C overnight. The secondary antibodies conjugated with horseradish peroxidase (HRP) were anti-rabbit (cat. no. NA934-1ML; Cytiva) and anti-mouse antibodies (cat. no. NA931-1ML; Cytiva) at a 1:2,000 dilution and incubated at room temperature for 1 h. Primary and secondary antibodies of GRHL2 were diluted by Can Get Signal™ (cat. no. NKB-101; Toyobo Life Science). The intensities of the bands were quantified with Fiji (v.2.9.0/1.53t) (https://imagej.net/software/fiji/) (32) or ImageJ (v.1.53k) (https://imagej.nih.gov/ij/index.html) (33), where values of intensities normalized to β-actin, AKT or ERK were shown below the corresponding band images in the figures.